Method and cell line for obtaining plasminogen activators

ABSTRACT

A method of increasing the yield of plasminogen activator from a plasminogen activator-producing cell line by adapting said cell line to grow in a suitable growth medium essentially free of fetal calf serum is disclosed. Also disclosed are cell lines capable of producing relatively large quantities of plasminogen activator, and a chemically defined growth medium suitable for adapting a plasminogen activator-secreting cell line to growth in the absence of fetal calf serum.

This application is a continuation-in-part of application Ser. No.601,868, filed Apr. 19, 1984 now abandoned.

BACKGROUND OF THE INVENTION

Plasminogen activator (PA) is a serine protease which exerts its actionthrough hydrolysis of the Arg₅₆₀ -Val₅₆₁ peptide bond in plasminogen,yielding the 2-chain plasmin molecule. Plasmin has a general proteolyticactivity and in the physiological environment of circulating blood itattacks mainly fibrin. Plasminogen activator plays a key role in thefibrinolytic system, which by lysing intra- or extra-vascular thrombi,clots, or fibrinous deposits, profoundly influences the incidence ofthromboembolic vascular disease and its outcome.

Plasminogen activators have been isolated from body fluids such as bloodand urine, and solid tissues of various histologic origin. Mammaliancell cultures are also known to produce plasminogen activators. Theremay be large differences in the amounts of plasminogen activatorproduced by cell lines derived from different tissues of the sameanimal, by cells of the same histologic type derived from cells of asingle type independently transformed with the same oncogenic agent.

There is an urgent need for obtaining cells which are capable ofproducing increased amounts of PA and establishing techniques whichwould improve the process of using animal cell culture to produce PA onan industrial scale.

DESCRIPTION OF PERTINENT ART

European patent application No. 113,319 A2 discloses human cell linescapable of proliferating in the absence of serum and othermacromolecular growth factors as well as the method utilized forproducing such cell lines. European patent application No. 112,174 A2describes a serum-free medium capable of growing a wide range ofsuspension and monolayer cells, said medium containing fetuin,transferrin and a substituted phosphatidyl-choline.

As taught in J. Biol. Chem., 256, 7035-7041 (1981), it is well-knownthat human cell lines of neoplastic origins, e.g. melanoma cells, canproduce substantial amounts of PA.

Malignant cell lines such as cervix carcinoma, larnyx carcinoma,epidermoid carcinoma of the oral cavity, colon and rectum carcinomacells have been shown to produce PA (see Molecular CellularBiochemistry, Vol. 15, No. 2, pages 149-153 (1977).

In Proc. Natl. Acad. Sci., Vol. 78, No. 9, pages 5673-5676 (1981), it isreported that two metastatic prostatic carcinoma cell lines, PC-3 andDU-145, have been grown in long-term culture in a defined medium free ofserum, hormones or growth factors.

A progressive loss of metastatic potential and tumorigenicity was notedfollowing serial passage in vitro of human epidermoid carcinoma.However, with regard to PA, high levels of production werewell-correlated with retention of tumorigenicity and metastasis, CancerResearch, 40, 2310-2315 (1980).

Barnes and Sato in "Growth of a Human Mammary Tumour Cell Line in aSerum-Free Medium" (Nature, October 1979) report the growth of the MCF-7cell line in a serum-free medium supplemented with insulin, transferrin,epidermal growth factor, prostagladin F₂α and cold-insoluble globulin.The synthetic nutrient medium used in this study was 1:1 mixture ofHam's F-12 and Dulbecco-modified Eagle's medium supplemented withantibiotics, sodium bicarbonate, HEPES and sodium selenite.

None of these references suggest or teach that the yield of PA fromPA-secreting cells can be increased by adapting such cells to grow in amedium which is essentially free of fetal calf serum.

SUMMARY OF THE INVENTION

The present invention is directed to a method of increasing the yield ofplasminogen activator (PA) obtained from a PA-producing cell line, and acell line capable of producing relatively large amounts of PA. Themethod involves growing PA-producing cells in a suitable growth mediumcontaining fetal calf serum (bovine serum) and adapting the cells togrow in said suitable growth medium essentially free of fetal calf serumby passing the cells through a series of said growth media containingdecreasing amounts of fetal calf serum.

A biologically pure tissue culture, capable of producing relativelylarge quantities of plasminogen activator is a human prostateadenocarcinoma, available from the American Type Culture Collection(ATCC) designated as Cell Repository Line (CRL)-1435. Other biologicallypure tissue cultures capable of producing relatively large quantities ofplasminogen activator when adapted according to the method of thepresent invention are ATCC CRL-1622 and ATCC CRL-1579.

DETAILED DESCRIPTION OF THE INVENTION

It is well-known that plasminogen activators can be produced by tumorcells in culture. Examples of tumor cells from mammals which can be usedin the present invention include melanoma, prostate, breast, colon,ovarian, pancreatic, cervical, rectal, endometrial and fibroblastictumor cell lines. Preferred cell lines are human carcinoma cell linessuch as melanoma, prostate, breast, colon, ovarian, pancreatic andendometrial. Suitable carcinoma cell lines are available fromdepositories such as American Type Culture Collection, 1201 ParklawnDrive, Rockville, MD 20852 or can be obtained from numerous researchersat universities, hospitals or research institutes. The identity of thecarcinoma cell line is not of critical importance; the present methodallows the amount of PA obtained from a PAproducing cell line to beincreased by as much as from about 5 to about 60 fold. As indicatedherein, a preferred high-yield producer of PA is a human prostateadenocarcinoma, ATCC CRL-1435. Other preferred biologically pure tissuecultures capable of producing relatively large quantities of plasminogenactivator when adapted according to the method of the present inventionare ATCC CRL-1622 and ATCC CRL-1579.

As used herein, the phrase "suitable growth medium" refers to a mediumsuch as described in detail hereinafter containing a mixture ofWaymouth's MB752/1, Dulbecco Minimal Essential Medium (MEM) and Ham'sF-12 medium in a ratio (by weight) of from about 1:1:1 to about 3:1:2,respectively.

The method involves selecting a parent carcinoma cell line whichproduces PA, and growing the cell line to confluency in a suitablegrowth medium which contains at the outset from about 5 to about 20percent fetal calf serum. After the cell line reaches confluency, thecells are removed, usually by trypsinization and subjected tosubculturing in a series of suitable growth media which containdecreasing amounts of fetal calf serum. The rate of removal of the fetalcalf serum can be easily determined by sequentially reducing the calfserum and examining the viability of the cell growth. For example, theparent cells can be grown in a suitable growth medium containingapproximately 10 percent fetal calf serum to confluency. The cells arethen removed and subcultured through at least one passage in a suitablegrowth medium containing approximately 5 percent fetal calf serum. Afterthe cells reach confluency, the cells can be removed again andsubcultured through at least one passage in a suitable growth mediumcontaining approximately 2.5 percent fetal calf serum. This procedurecan be repeated until the suitable growth medium used is essentiallyfree of fetal calf serum. In the present invention, sequential reductionby a 2-fold factor has been found to be a preferred method.

It has been found that various combinations of commercially availablegrowth media are suitable for use in the present invention. However, asthe concentration of fetal calf serum is decreased, the suitable growthmedium used in the method of the present invention must becorrespondingly supplemented with the following growth factors: fetuin,bovine serum albumin, insulin, transferrin, 5α-dihydrotestosterone anddexamethasone. The point during the sequential reduction of fetal calfserum at which these growth factors must be added may be readilydetermined by one skilled in the art and will vary depending on factorssuch as the type of cell lines being cultured and the specificconditions under which the culturing is being carried out. Suffice it tosay that the growth factors must be added at a point when cell viabilitymight otherwise be threatened due to the decreased concentration offetal calf serum; however, when the suitable culture medium contains asufficient concentration of fetal calf serum to sustain the growth ofthe cell line, addition of the growth factors noted above is unnecessaryand may lead to subsequent difficulty in adapting the cell line to growin the suitable serum-free growth medium.

A suitable growth medium for use as described herein can be obtained bymixing together Waymouth's MB752/1 medium, Dulbecco MEM and Ham's F-12medium, commercially available from GIBCO Laboratories, Grand Island,New York. The ratio (by weight) can range from 1:1:1 to 3:1:2,respectively.

A preferred medium, hereinafter referred to as "SYC" medium, is composedof a 1:1:1 (by weight) mixture of Waymouth's MB752/1, Dulbecco MEM, andHam's F-12, respectively. It has been observed that cell growth isoptimized when the suitable growth medium used is SYC medium.

In the total absence of Waymouth's MB752/1, cell growth is greatlyretarded. These media have the compositions as set forth in Table I.

                                      TABLE I                                     __________________________________________________________________________    (Concentration of each component in mg/l)                                                 Waymouth MB752/1                                                                         Ham's F-12                                                                          Dulbecco MEM                                                                           SYC Medium                              __________________________________________________________________________    CaCl.sub.2 (anhydrous)                                                                    90.61      33.22 200      107.94                                  CuSO.sub.4.5H.sub.2 O                                                                     --         0.00249                                                                             --       0.00083                                 Fe(NO.sub.3).sub.3.9H.sub.2 O                                                             --         --    0.1      0.033                                   FeSO.sub.4.7H.sub.2 O                                                                     --         0.834 --       0.278                                   KCl         150        223.6 400      257.87                                  KH.sub.2 PO.sub.4                                                                         80         --    --       26.67                                   MgCl.sub.2  112.56     57.22 --       56.59                                   MgSO.sub.4  97.67      --    97.67    65.11                                   NaCl        6000       7599  6400     6666.33                                 Na.sub.2 HPO.sub.4                                                                        300        142.04                                                                              --       147.35                                  NaH.sub.2 PO.sub.4.H.sub.2 O                                                              --         --    125      41.67                                   ZnSO.sub.4.7H.sub.2 O                                                                     --         0.863 --       0.288                                   L-Alanine   --         8.9   --       2.97                                    L-Arginine HCl                                                                            75         211   84       123.33                                  L-Asparagine.H.sub.2 O                                                                    --         15.01 --       5.0                                     L-Aspartic acid                                                                           60         13.30 --       24.43                                   L-Cystine.2HCl                                                                            19.55      --    62.57    27.37                                   L-Cysteine HCl.H.sub.2 O                                                                  100.26     35.12 --       45.13                                   L-Glutamic acid                                                                           150        14.7  --       54.9                                    L-Glutamine 350        146   584      360                                     L-Glycine   50         7.5   30       29.17                                   L-Histidine HCl.H.sub.2 O                                                                 164.1      20.96 42       75.69                                   L-Isoleucine                                                                              25         3.94  105      44.65                                   L-Leucine   50         13.10 105      56.03                                   L-Lysine HCl                                                                              240        36.5  146      140.83                                  L-Methionine                                                                              50         4.48  30       28.16                                   L-Phenylalanine                                                                           50         4.96  66       40.32                                   L-Proline   50         34.5  --       28.17                                   L-Serine    --         10.5  42       17.50                                   L-Threonine 75         11.9  95       60.63                                   L-Tryptophane                                                                             40         2.04  16       19.35                                   L-Tyrosine (Na salt)                                                                      57.66      7.78  103.79   56.41                                   L-Valine    65         11.70 94       56.9                                    Ascorbic acid                                                                             17.5       --    --       5.83                                    Biotin      0.02       0.0073                                                                              --       0.0091                                  Ca pantothenate                                                                           1.0        0.48  4.0      1.83                                    Choline Cl  250        13.96 4.0      89.32                                   Folic acid  0.4        1.30  4.0      1.9                                     i-Inositol  1.0        18.0  7.2      8.73                                    Nicotinamide                                                                              1.0        --    4.0      1.67                                    Niacinamide --         0.037 --       0.012                                   Pyridoxine HCl                                                                            1.0        0.062 4.0      1.69                                    Riboflavin  1.0        0.038 0.40     0.48                                    Thiamine HCl                                                                              10.0       0.34  4.0      4.78                                    Vit. B.sub.12                                                                             0.2        1.36  --       0.52                                    D-Glucose   5000       1802  1000     2600                                    Glutathione (reduced)                                                                     15         --    --       5                                       Hypoxanthine (Na salt)                                                                    29         4.77  --       11.26                                   Phenol red  10         1.2   15       8.73                                    Thymidine   --         0.73  --       0.24                                    Linoleic acid                                                                             --         0.084 --       0.028                                   Lipoic acid --         0.21  --       0.07                                    Putrescine.2HCl                                                                           --         0.161 --       0.054                                   Na pyruvate --         110   110      73.33                                   __________________________________________________________________________

As described herein, as the concentration of fetal calf serum isserially decreased in each passage, it becomes necessary to supplementthe suitable growth medium with from about 50-150 mg/l fetuin; fromabout 50-150 mg/l bovine serum albumin; from about 5-10 mg/l insulin;from about 5-40 mg/l transferrin; from about 50-200 μg/l5α-dihydrotestosterone; and from about 50-200 μg/l dexamethasone. It hasbeen observed that insulin and transferrin are critical growth factorswithout which the cells will die after one passage where all growthfactors would otherwise have been added to supplement the suitablegrowth medium.

The following examples are provided as a means of illustrating thepresent invention and should not be construed as a limitation thereon.

EXAMPLE I

Following receipt from the depository, a human prostate adenocarcinoma,ATCC CRL-1435, was stabilized by growing to confluency in Earle'sMinimal Essential Medium (MEM) commercially available from GIBCO. Thisprocedure was carried out by placing a 25 ml portion of MEM plus 10percent fetal calf serum in a 175 ml culture flask, maintained at 37° C.in a 5 percent CO₂ atmosphere, and adding 5×10⁵ of the aboveadenocarcinoma cells. The parent cells were removed from the flask byadding a 5 ml portion of 0.25 percent trypsin.

The parent cells from the above stabilization procedure were then addedto a previously prepared quantity of SYC medium containing 10 percentfetal calf serum and were maintained at 37° C. The cells reachedconfluency in approximately one week after which they were removed bytrypsinization as described above and were added to a quantity of SYCmedium containing 5 percent fetal calf serum. The cells were againmaintained at 37° C. until they reached confluency. This procedure wasrepeated twice using SYC medium containing 2.5 percent and 1.25 percentfetal calf serum, respectively. At the 1.25 percent fetal calf serumconcentration, the SYC medium was supplemented with fetuin (75 mg/l),bovine serum albumin (75 mg/l), insulin (8 mg/l), transferrin (25 mg/l),5.sub.α -dihydrotestosterone (100 μg/l) and dexamethasone (100 μg/l).The cells reached confluency in approximately ten days.

The cells were then passaged into SYC medium containing the same growthfactors as in the 1.25 percent fetal calf serum passage, except that nofetal calf serum was present. The cells reached confluency inapproximately one week.

The above cells were then passaged ten times through SYC medium(containing the same growth factors as above), free of fetal calf serum,until the cell line stabilized. Determination of stabilization wasachieved by observing the same growth pattern during each passage andthe amount of PA produced. After ten passages, the biologically purealtered confluent cells, designated PC-3f, and deposited with ATCChaving accession number CRL-8539 were assayed for PA production by thefollowing procedure (as described in Progress in Chemical Fibrinolysisand Thrombolysis, Vol. 3, pages 315-322; 1978).

A 100 μl portion of culture fluid (obtained after confluency) wasincubated with 100 μl of human plasminogen (0.33 mg/ml) for 15 minutesat 37° C. after which 250 μl of Tris buffer (0.1 M Tris; 0.2 M NaCl; pH7.4) and 250 μl of 1 mM tripeptide substrate(Valleu-lys-p-nitro-anilide; Kabi, Stockholm, Sweden) were added. Thismixture was incubated for an additional 3 minutes after which thereaction was stopped by the addition of 100 μl of 50% acetic acid. Theabsorbance of the reaction mixture was read at 405 nm and was thencompared to a urokinase standard curve generated with commerciallyavailable urokinase (Calbiochem., La Jolla, CA) using the same assayprocedure. This assay technique allows quantitation of the PA producedby the cells by measuring the quantity of plasmin produced by activationof the plasminogen from the PA present in the culture fluid.

The test results are set forth in Table II. The amount of PA produced bythe cells is expressed in CTA units per ml of culture fluid asstipulated by the Committee on Thrombolytic Agents of the National HeartInstitute, as described in Thromb. Diath. Haemor., 21, page 259 (1969).As a control, the PA activity of the parent cell line (ATCC-1435) wasalso determined.

                  TABLE II                                                        ______________________________________                                        (PA activity expressed in CTA Units/ml)                                       Cell Line      48 Hours  72 Hours  96 Hours                                   ______________________________________                                        ATCC CRL-1435  11.5      18.5      Died                                       PC-3f (ATCC CRL-8539)                                                                        28.5      45.0      70.4                                       ______________________________________                                    

As shown in the above test results, passage of the parent cell linethrough serum-free medium produced a cell line which yielded up to 70.4CTA Units of PA, as compared to a maximum of 18.5 CTA Units of PAproduced by the parent cell line, an increase of approximately 4-fold.Advantageously, the altered cell line was viable for an additional 24hours.

EXAMPLE II

The procedure described in Example I was repeated using as a parent cellline a human pancreatic carcinoma cell line, ATCC-1420. The biologicallypure altered cells obtained by the procedures described herein weredesignated as PaCa-2f and were deposited with ATCC having accessionnumber ATCC CRL-8725. Test results obtained are summarized in Table IIIbelow:

                  TABLE III                                                       ______________________________________                                        (PA activity expressed in CTA Units/ml)                                       Cell Line       48 Hours  72 Hours  96 Hours                                  ______________________________________                                        ATCC-1420       2.5        7.5      Died                                      PaCa-2f (ATCC CRL-8725)                                                                       5.4       15.0      25.2                                      ______________________________________                                    

As shown in the test results, passage of the parent cell line throughserum-free medium produced a cell line which yielded PA up to 25.2 CTAUnits, an increase of approximately 3-fold, as well as a 24 hourincrease in viability.

EXAMPLE III

The procedure described in Example I was repeated using as a parent cellline a mouse melanoma cell line, B-16, available from Jackson Lab., BarHarbor, Maine. The biologically pure altered cells obtained by theprocedure described herein were designated as B-16_(f). Test resultsobtained are summarized in Table IV below:

                  TABLE IV                                                        ______________________________________                                        (PA activity expressed in CTA Units/ml)                                       Cell Line                                                                              24 Hours 48 Hours   72 Hours                                                                             96 Hours                                  ______________________________________                                        B-16     0.70     1.20       Died   --                                        B-16.sub.f                                                                             2.30     2.80       3.0    3.2                                       ______________________________________                                    

The biologically pure altered cell line showed a 3-fold increase in PAproduction at 24 hours and a 2-fold increase in PA production at 48hours. Further, the altered cell line was viable for up to 96 hours.

EXAMPLE IV

The procedure described in Example I was repeated using as a parent cellline ATCC CRL-1622, an endometrial adenocarcinoma cell line availablefrom the ATCC. The biologically pure altered cells obtained by themethod of the present invention were designated as KLE_(f) and weredeposited with ATCC having accession number ATCC CRL-8726. PA activitywas measured at 48 hours after the cells reached confluency and theresults obtained are shown in Table V.

                  TABLE V                                                         ______________________________________                                        (PA activity expressed in CTA Units/ml)                                       Cell Line          48 Hours                                                   ______________________________________                                        ATCC CRL-1622      0.05                                                       KLE.sub.f (ATCC CRL-8726)                                                                        3.03                                                       ______________________________________                                    

As shown in Table V, the altered cell line, KLE_(f), showed over a60-fold increase in PA production over the parent cell line at 48 hours.

EXAMPLE V The procedure described in Example I was repeated using as aparent cell line an amelanotic melanoma cell line available from ATCCunder accession number ATCC CRL-1579. The biologically pure alteredcells obtained by the method of the present invention were designatedML_(f) and were deposited with ATCC having accession number ATCCCRL-8724. PA activity was measured at 48 hours after the cells reachedconfluency and the results obtained are shown in Table VI.

                  TABLE VI                                                        ______________________________________                                        (PA activity expressed in CTA Units/ml)                                       Cell Line          48 Hours                                                   ______________________________________                                        ATCC CRL-1579      0.sup.a                                                    ML.sub.f (ATCC CRL-8724)                                                                         0.07                                                       ______________________________________                                         .sup.a Activity level was below detection limits of the assay.           

What is claimed is:
 1. A method for increasing the yield of plasminogenactivator obtained from plasminogen activator-producing mammalian tumorcells which comprises the steps of:(a) growing said cells in a suitablegrowth medium supplemented with fetal calf serum, said suitable growthmedium composed of a mixture of Waymouth's MB752/1, Dulbecco MinimalEssential Medium and Ham's F-12 medium in a ratio (by weight) of fromabout 1:1:1 to about 3:1:2, respectively,; (b) passing said cellsthrough a series of said suitable growth media containing decreasingamounts of fetal calf serum, said media supplemented with the growthfactors fetuin, bovine serum albumin, insulin, transferrin,5α-dihydrotestosterone and dexamethasone; (c) growing said cells toconfluency in said suitable growth medium essentially free of fetal calfserum and supplemented with said growth factors, said cells producingplasminogen activator at levels greater than that of the initialplasminogen activator-producing cells; and (d) recovering therefrom saidplasminogen activator.
 2. The method of claim 1 wherein the plasminogenactivator-producing cells are mammalian tumor cells selected from thegroup consisting of melanoma, prostate, breast, colon, ovarian,pancreatic, cervical, rectal, endometrial and fibroblastic tumor cells.3. The method of claim 2 wherein the mammalian tumor cells are humancarcinoma cell lines selected from the group consisting of melanoma,prostate, breast, colon, ovarian, pancreatic and endometrial cell lines.4. The method of claim 3 wherein the tumor cell line is a human prostateadenocarcinoma.
 5. The method of claim 1 wherein the suitable growthmedium is an SYC medium composed of a 1:1:1 by weight mixture ofWaymouth's MB752/1, Dulbecco MEM and Ham's F-12, respectively.
 6. Themethod of claim 5 wherein the fetuin is present in a concentration offrom 50-150 milligrams per liter; the bovine serum albumin is present ina concentration of from 50-150 milligrams per liter; the insulin ispresent in a concentration of from 5-10 milligrams per liter; thetransferrin is present in a concentration of from 5-40 milligrams perliter; the 5α-dihydrotestosterone is present in concentration of from5-200 micrograms per liter; and the dexamethasone is present in aconcentration of from 50-200 micrograms per liter.
 7. The method ofclaim 6 wherein the fetuin is present in a concentration of 75milligrams per liter; the bovine serum albumin is present in aconcentration of 75 milligrams per liter; the insulin is present in aconcentration of 8 milligrams per liter; the transferrin is present in aconcentration of 25 milligrams per liter; the 5α-dihydrotestosterone ispresent in a concentration of 100 micrograms per liter; and thedexamethasone is present in a concentration of 100 micrograms per liter.8. The method of claim 1 wherein the concentration of fetal calf serumis serially decreased by a 2-fold factor through each of said passages.9. A method for increasing the yield of plasminogen activator obtainedfrom human prostate adenocarcinoma cell line ATCC CRL-1435 whichcomprises the steps of:(a) growing said cell line in a suitable growthmedium supplemented with fetal calf serum, said suitable growth mediacomposed of a mixture of Waymouth's MB752/1, Dulbecco Minimal EssentialMedium and Ham's F-12 Medium in a ratio (by weight) of from about 1:1:1to about 3:1:2, respectively; (b) passing said cell line through aseries of said suitable growth media containing decreasing amounts offetal calf serum, said media supplemented with the growth factorsfetuin, bovine serum albumin, insulin, transferrin,5α-dihydrotestosterone and dexamethasone; (c) growing said cell line toconfluency in said suitable growth medium essentially free of fetal calfserum and supplemented with said growth factors, said cell lineproducing plasminogen activator at levels greater than that of theinitial plasminogen activator-producing cell line; and (d) recoveringtherefrom said plasminogen activator.
 10. The method of claim 9 whereinsaid suitable growth medium is an SYC medium composed of a 1:1:1 byweight mixture of Waymouth's MB752/1, Dulbecco MEM and Ham's F-12,respectively.
 11. The method of claim 10 wherein the fetuin is presentin a concentration of from 50-150 milligrams per liter; the bovine serumalbumin is present in a concentration of from 50-150 milligrams perliter; the insulin is present in a concentration of from 5-10 milligramsper liter; the transferrin is present in a concentration of from 5-40milligrams per liter; the 5α-dihydrotestosterone is present in aconcentration of from 50-200 micrograms per liter; and the dexamethasoneis present in a concentration of from 50-200 micrograms per liter. 12.The method of claim 11 wherein the fetuin is present in a concentrationof 75 milligrams per liter; the bovine serum albumin is present in aconcentration of 75 milligrams per liter; the insulin is present in aconcentration of 8 milligrams per liter; the transferrin is present in aconcentration of 25 milligrams per liter; the 5α-dihydrotestosterone ispresent in a concentration of 100 micrograms per liter; and thedexamethasone is present in a concentration of 100 micrograms per liter.13. The method of claim 9 wherein the concentration of fetal calf serumis serially decreased by a 2-fold factor through each of said passages.14. A biologically pure culture of a plasminogen activator-secretingcell line designated as PC-3f having ATCC accession number CRL-8539. 15.A biologically pure culture of a plasminogen activator-secreting cellline designated as PaCa-2f having ATCC accession number CRL-8725.
 16. Abiologically pure culture of a plasminogen activator-secreting cell linedesignated as KLE_(f) having ATCC accession number CRL-8726.
 17. Abiologically pure culture of a plasminogen activator-secreting cell linedesignated as ML_(f) having ATCC accession number CRL-8724.